Human heart rate monitors (HRMs) are frequently used in equine studies to measure heart rate (HR) and interbeat intervals (IBIs). However, to date, the most commonly used HRM (the Polar® system) in horses has not been validated against simultaneously recorded electrocardiogram (ECG) signals during a range of ambulatory conditions. Polar® S810i and ECG IBIs were simultaneously recorded from six horses under three conditions commonly included in behavioural observation: standing at rest, loose in the stable and at liberty in a field. Following recording, Polar® IBI data were corrected for error processing in cardiac data. Corrected and uncorrected Polar® data were then compared with simultaneously recorded ECG data using a variety of commonly measured time and frequency domain parameters (e.g. HR variability (HRV)). Polar® data collected while horses were stabled or in the field were significantly different from ECG data, even following correction of the data, and therefore, it may not be possible for the two systems to be used interchangeably. This study indicates the need for caution while using Polar® S810i for collecting HRV data, unless horses are stationary, and even when the IBI data are corrected for measurement error.